Typically, conventional fluorescent dyes (e.g., fluorescein, rhodamine, phycoerythrin, and the like) are used for labeling microspheres. These conventional fluorescent dyes typically have an excitation spectrum that may be quite narrow; hence, it is often difficult to find a wave-length spectrum of light suitable for simultaneously exciting several different fluorescent labels (e.g., differing in color of fluorescence emission). However, even when a single light source is used to provide a excitation wave-length spectrum (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different species (e.g., differing in color) of fluorescent dyes to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent dyes are susceptible to photobleaching which limits the time in which a fluorescent signal can be detected, and limits time-resolved fluorescence (fluorescent signal integration over time). Additional limitations of fluorescent dyes include fluorescence quenching, and shifts in fluorescence emission spectra, depending on the environment in which dyes are excited.
Fluorescent nanocrystals comprising either semi-conductor nanocrystals or doped metal oxide nanocrystals have been reported to resist photobleaching, share an excitation wavelength spectrum, and are capable of emitting fluorescence of high quantum yield and with discrete peak emission spectra. However, these nanocrystals lack sufficient solubility in aqueous-based environments required in fluorescence-based biological assays; i.e., in aqueous-based environments, the nanocrystals interact together in forming aggregates, which leads to irreversible flocculation of the nanocrystals. As disclosed in detail in U.S. application Ser. Nos. 09/372,729 and 09/577,761 (the disclosures of which are herein incorporated by reference), functionalized fluorescent nanocrystals comprise fluorescent nanocrystals which have been functionalized by the addition of a plurality of molecules; and preferably, the molecules are selected from an amino acid, a carboxylic acid, and a combination thereof. A plurality of these molecules, when operably bound to a fluorescent nanocrystal, functionalizes the fluorescent nanocrystal to become water-soluble, as well as provides reactive functionalities which may be used to operably bind one or more molecules of affinity ligand. Functionalized fluorescent nanocrystals, comprising affinity ligand operably bound thereto, may be placed in contact with a sample being analyzed in a biological assay for the presence or absence of a substrate for which the affinity ligand has binding specificity. Contact, and subsequent binding, between affinity ligand of the functionalized fluorescent nanocrystals and the substrate, if present in the sample, results in a complex comprising the functionalized fluorescent nanocrystals-substrate which can emit a detectable fluorescence signal for quantitation, visualization, or other form of detection.
However, current methods of functionalizing fluorescent nanocrystals with affinity ligand suffer from a serious drawback. Even with a high efficiency of operably binding functionalized fluorescent nanocrystals with affinity ligand, the coupling reaction may result in a significant number of functionalized fluorescent nanocrystals which do not become operably bound to affinity ligand, and molecules of affinity ligand which do not become operably bound to functionalized fluorescent nanocrystals ("free affinity ligand"); a phenomenon referred to as "failed coupling". In a biological assay in which an amount of substrate is to be detected using a detection reagent which contains both functionalized fluorescent nanocrystals having affinity ligand operably bound thereto, and free affinity ligand, free affinity ligand can compete with the affinity ligand of functionalized fluorescent nanocrystals for binding the substrate. Particularly in instances when a minute amount of substrate is present, the effect of binding substrate by free affinity ligand in the detection reagent can lead to an undesirable and significant loss of sensitivity in the assay. In contrast, functionalized fluorescent nanocrystals which are not operably bound to affinity ligand will be washed away from a detection system, and hence, do not pose a significant problem if present in a detection reagent.
Thus, there remains a need for a process of purifying functionalized fluorescent nanocrystals substantially free of free affinity ligand that may be present after a step of operably binding affinity ligand to the functionalized fluorescent nanocrystals. Also needed is a purification method which is simple, uses relatively few reagents, and provides functionalized fluorescent nanocrystals having affinity ligand operably bound thereto at a high level of purity for use in fluorescence-based detection systems.